Manipulation of microRNA for CNS delivery: implication to treatment of neurological LSD

用于中枢神经系统递送的 microRNA 操作：对神经性 LSD 治疗的影响

基本信息

批准号：
10201374
负责人：
Dao Pan
金额：
$ 55.65万
依托单位：
CINCINNATI CHILDRENS HOSP MED CTR
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-15 至 2022-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10201374
关键词：
3&apos Untranslated Regions Adult Allogenic Alzheimer&apos s Disease Behavior Behavioral Biodistribution Blood - brain barrier anatomy Blood Circulation Brain Brain Diseases Capillary Endothelial Cell Capsid Cell Therapy Cells Childhood DNA cassette Data Dependovirus Development Disease Down-Regulation Drug Delivery Systems Enzymes Epigenetic Process Erythroid Excision Foundations Frequencies Functional disorder Future Gene Transfer Genetic Glycosaminoglycans Goals Hematopoietic Stem Cell Transplantation Histopathology Human Hybrids IGF Type 2 Receptor IGF2 gene IGF2R gene Individual Inherited Injections Intravenous Knock-out L-Iduronidase Live Birth Luciferases Lysosomal Storage Diseases Lysosomes Mediating Medical Messenger RNA Metabolic Metabolic Diseases MicroRNAs Modeling Modification Mucopolysaccharidosis I Mucopolysaccharidosis I H Mus Mutagenesis Nerve Nerve Degeneration Neurodegenerative Disorders Neurologic Organ Parkinson Disease Pathologic Pathway interactions Patients Penetration Periodicals Play Porifera Production Proteins Public Health Recombinants Reporter Repression Resolution Role Severities Site Specificity System Therapeutic Transfection Translations Up-Regulation Variant Vascular Endothelial Cell Visceral adeno-associated viral vector base brain cell caspase 14 cell type cerebral capillary cerebral microvasculature correctional system design enzyme replacement therapy enzyme therapy improved in vivo inhibitor/antagonist macromolecule mouse model nervous system disorder novel novel therapeutics postnatal period preclinical evaluation promoter prototype receptor receptor downregulation receptor expression therapeutic enzyme trafficking transcytosis transgene expression

项目摘要

Abstract Lysosomal storage disorders (LSDs) are a group of inherited metabolic diseases characterized by a dysfunction in lysosomes, with cumulative frequency of 1 in 7000 live births (although individually rare). Over 2/3 of LSD patients present an involvement of the central nerve system (CNS) with a broad spectrum of severity (nLSD), which makes LSDs the most common cause of pediatric neurodegenerative disease. Allogeneic hematopoietic stem cell transplantation (HSCT) or enzyme replacement therapy (ERT) by periodical injection of recombinant enzyme are main treatment options for nLSD. However, they are largely unsuccessful in reversing neurological complications due to the poor penetration of the enzymes across the blood-brain-barrier (BBB) to the CNS, a major obstacle in treating nLSD. The cation-independent mannose-6-phosphate receptor (M6PR, also called IGF2R) plays a critical role in lysosomal enzyme trafficking and intercellular transfer of most lysosomal enzymes, which is essential for metabolic cross-correction in treating LSDs. Developmental decline of M6PR on the BBB during early postnatal period in mouse and human has been documented, which is attributable to the lack of CNS enzyme delivery. Using a dual luciferase reporter system with site-mutagenesis, we have recently discovered that microRNA-143 (miR143) modulate M6PR protein levels on BBB-forming brain capillary endothelial cells (BrMV) by targeting to 3' untranslated region of M6PR mRNA. Using a mouse model of Hurler syndrome (severe mucopolysaccharidosis type I, MPS I), which is caused by the deficiency of α-L-iduronidase (IDUA), we further demonstrated functional rescue of M6PR-mediated IDUA transfer in the brain of double- knockout (MPS/miR-143KO) mice with long-term CNS therapeutic benefits, as well as in human vascular endothelial cells by down-regulation of miR-143 with miR-143-sponge sequences. The data provide strong scientific premise for the development of a novel CNS-targeted approach that would be applicable in treating many neurologic LSDs involving M6PR pathway, or in delivering brain therapeutics that can adapting M6PR- mediated transcytosis pathway. In this proposal, we aim to develop a novel adeno-associated viral vector (AAV)- based translatable platform to “restore” M6PR pathway on mature BBB for advanced delivery of therapeutic enzymes into the CNS with 3 aims, including developing optimal artificial miR143 inhibitor (143in) and expression cassette(s) for efficient and targeted reduction of miR143 on BrMV (aim 1), examination of biodistribution and “off-target” expression and effects in mice with AAV-143in delivery (aim 2), as well as preclinical evaluation of BBB-targeted AAV/miR143in in correcting CNS abnormalities in MPS I mice by enzyme therapy derived from genetically modified erythroid/ megakaryocytic lineages (aim 3). The impact of the study is driven by the unmet medical need for efficient treatment of inherited nLSDs AND the major limitation of drug-delivery across the BBB.

抽象的溶酶体贮积症（LSD）是一组以功能障碍为特征的遗传性代谢疾病在溶酶体中，累积频率为每 7000 名活产婴儿中就有 1 人（尽管个别罕见）超过 LSD 的 2/3。患者出现中枢神经系统（CNS）受累，严重程度广泛（nLSD），这使得 LSD 成为儿童神经退行性疾病的最常见原因。干细胞移植（HSCT）或酶替代疗法（ERT）通过定期注射重组酶是 nLSD 的主要治疗选择，然而，它们在逆转神经功能方面基本上不成功。由于酶穿过血脑屏障（BBB）到达中枢神经系统的渗透性差而导致并发症，治疗 nLSD 的主要障碍是阳离子非依赖性甘露糖 6 磷酸受体（M6PR，也称为 IGF2R）在溶酶体运输酶和大多数溶酶体酶的细胞间转移中起着关键作用，这对于治疗 LSD 的代谢交叉校正至关重要。 BBB 上 M6PR 的发育下降。在小鼠和人类的产后早期已有记录，这是由于缺乏我们最近使用具有位点诱变的双荧光素酶报告系统进行中枢神经系统酶递送。发现 microRNA-143 (miR143) 调节 BBB 形成脑毛细血管中的 M6PR 蛋白水平使用 Hurler 小鼠模型，通过靶向 M6PR mRNA 的 3' 非翻译区来检测内皮细胞 (BrMV)。由 α-L-艾杜糖醛酸酶缺乏引起的综合征（I 型严重粘多糖贮积症，MPS I）（IDUA），我们进一步证明了 M6PR 介导的 IDUA 转移在双脑大脑中的功能性拯救敲除 (MPS/miR-143KO) 小鼠具有长期中枢神经系统治疗益处，以及对人类血管的益处 miR-143-sponge 序列通过下调 miR-143 来抑制内皮细胞的生长。开发适用于治疗的新型中枢神经系统靶向方法的科学前提许多涉及 M6PR 通路的神经LSD，或提供可以适应 M6PR 的脑部治疗药物在本提案中，我们的目标是开发一种新型腺相关病毒载体（AAV）- 基于可翻译平台来“恢复”成熟 BBB 上的 M6PR 通路，以实现治疗的高级传递将酶引入中枢神经系统有 3 个目标，包括开发最佳的人工 miR143 抑制剂 (143in) 和表达用于有效和有针对性地减少 BrMV 上的 miR143（目标 1）、检查生物分布和 AAV-143 递送小鼠中的“脱靶”表达和影响（目标 2），以及临床前评估 BBB 靶向 AAV/miR143in 通过酶疗法纠正 MPS I 小鼠中枢神经系统异常转基因红细胞/巨核细胞谱系（目标 3）该研究的影响是由未满足的问题驱动的。有效治疗遗传性 nLSD 的医疗需求以及跨血脑屏障药物输送的主要限制。